Ba11gladesh ]. Fish. Res., 5(1), 2001: 53-58 Bacterial load in pond water and different organs of a Indian major carp Cirrhinus mrigala Ham. A.N.H. Banu* 1, M.A. Islam and M.B.R. Chowdhury 1 Department offish. Biology & Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 3 Department of Aquaculture, BAU, Mymensingh 2202 ' 1 Present & corresponding address: Bangladesh Fisheries Research Institute, Mymensingh 2201, Bangladesh Abstract During the study period (August, 1993 ro July,l994) the mean bacterial load in surface water was found to vary from 1.39 xl0 5 (July'94) ro 3.llxl0 7 CFU/ml (September'93), while that of botrom water ranged from l.olxl0 6 (May'94) to 5.90xl0 7 CFU/ml (Ocrober '93).The mean total number of bacterial load in body slime, liver and kidney was found to vary from 0.58xl0 3 (July'94) to 2.37xl0 7 CFU/g (March'94),from 0.22xl0 3 (July'94)to 9.64xl0 6 CFU/g (March'94) from O.l5xl0 3 (July'94) to 9.36xl0 6 CFU/g (March'94), respectively. Bacterial load in slime was significantly correlated with bacterial load in liver, bacterial load in slime was significantly correlated with bacterial load in kidney and bacterial load in liver was significantly correlated with bacterial load in kidney. Key words: Bacteria, C. mrigala, Pond water Introduction Wide ranges of bacterial flora are abundant in water and associated with fish specially, with the bottom living fishes. Effective water management in fish pond is one of the important factor contributing to the success of fish culture. Environment plays a crucial role in disrupting the balance between the host and the pathogen. Bacteria in aquatic systems, specially, freshwater systems have been employed as an index of abundance of the microbial community Horseley (1973) investigated relationship between the bacterial flora of salmon and its environment and observed that the bacteria of skin were similar with the bacteria in water. Frazier and Westhoff (1978) stated that the bacterial flora of living fish depends upon the microbial content of the water in which they live. Study of aquatic bacteria associated with fish is very limited in Bangladesh. Few attempts have been taken in order to assess the bacterial population in aquatic environment and their involvement in causing disease in fish Cirrhinus mrigala H. is one of the cultured fish in Bangladesh and considered as one ofthe important components in our poly-culture system. Considering, the above reasons the present study was undertaken to investigate the bacterial load in pond water and different organs of the bottom living fish, Cirrhinus mrigala H.
A. N.H. Banu eta/. Materials and methods Sampling: Fish samples (Length26.37cm to 35.62cm) of mrigal, Cirrhinus mrigala H. were collected once a month from the selected experimental pond of the Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh for period of 12 months as shown in Table 1. Water samples was collected also from the same pond once a month during the study period. Water samples were collected in sterilized reagent bottles both from the surface and bottom levels for bacterial population study as well as for studying the physico- chemical parameters. Water study: Surface and bottom water of the pond was carried out followed the pour and spread plate method as stated in Standard Methods for the examination of Water and waste water (Lenore et al.l989). Petri dishes were set out, two plates per dilution were tested. Each dilution (O.lml) was pipetted into the center ofthe petri dishes using a fresh pipette for each dilution. With a sterilized glass rod the content was spread as quickly as possible and the medium was allowed to set. Then the petri dishes were inverted and kept in incubator at 25 C for 24-48hr.The plates having 30-300 colonies were counted for viable count. The colony or viable count /ml was calculated by multiplying the average number of colonies per countable plate by the reciprocal of the dilution. Body slime: Fish samples collected for investigation were killed by a light hurt on the neck region and then the slimes were collected by scrapping with a sterile scalpel and were kept in a sterile weighing boat which were pre- weighed. Necessary dilution were made using physiological saline (0.85% NaCL) following the ten fold dilution method, Thus the samples were ready for inoculation in the culture medium.(tsa,oxoid). Liver: The body surface of fish samples were disinfected with 70% ethyl alcohol and their abdomen opened by aseptic dissection and then the liver was taken out carefully with the help of sterilized forceps (Austin and Al-Zaharanil988, Kabatal985). The liver samples after being weighed individually in a sterile weighing boat and was homogenized for preparation of suspension in physiological saline. Thereafter, the suspension was diluted with sterile physiological saline for inoculation on the culture media (TSA,Oxoid). Kidney: The kidney samples were prepared following the same procedure as done for the preparation ofliver. Results and discussion Average length and weight of fish samples were presented in Table 1. Length of fish ranged from 27.24cm to 35.62cm, while weight varied from 242.62g to 431.40g. The physico- chemical parameter of pond water is shown in Table 2. It was observed that the 54
Bacteria of C. mrigala physico- chemical parameter did not show any significant variation from one month to another. The mean total bacterial load of surface water during August, 1993 to July'94 was found to vary from 1.39xl0 5 CFU/ml(July'94) to 3.llxl0 7 CFU/ml(September'93) while that in case of bottom water ranged from l.olxl0 6 CFU/ml(May'94) to 5.06xl0 8 CFU/ml(September' 93)(Table 3).The bacterial load in case of surface water was found to be higher from August'93 to March'94 then it became lower from April'94 to July'94 while that in case of bottom water was found to fluctuate irregularly. Table 1. Average length and body weight of fish sample as recorded from August'93 to July'94 Month Length (em) Weight (g) 1st phase Aug.'93 30.32 304.90 Sep.'93 30.88 330.67 Oct.'93 32.68 347.60 Nov.'93 34.53 408.05 Dec.'93 33.00 346.00 Jan.'94 34.55 378.50 Feb.'94 33.95 367.00 Mar.'94 35.25 415.00 Apr.94 35.17 377.00 May.'94 35.62 431.40 June.'94 27.24 242.62 Jul.'94 27.36 288.60 LSD (0.1%) 2.35 71.55 Data presented are average of ten fish. Table 2. Monthly variation in physico- chemical parameters of the pond water during August'93 to July'94 Month Temp.( 0 C) ph DO (mg/1) Hardness Ammonia (mg/1) (mg/1) 1st phase Aug.'93 33.0 8.5 8.5 32.0 0.010 Sep.'93 29.7 7.2 8.5 32.0 0.001 Oct.'93 29.5 7.2 8.0 24.0 0.001 Nov.'93 29.7 7.0 8.5 24.0 0.001 Dec.'93 27.0 7.5 8.0 25.0 0.010 Jan.'94 21.3 8.0 7.5 27.0 0.100 Feb.'94 22.7 8.5 8.0 28.0 0.010 Mar.'94 26.3 8.0 7.2 40.0 0.010 Apr.94 29.6 8.0 7.5 27.0 0.001 May.'94 27.9 7.5 7.0 35.0 0.010 June.'94 32.4 7.5 7.5 40.0 0.020 Jul.'94 35.3 8.0 Data presented are average of four replications 7.5 40.0 1.020 55
A. N.H. Banu eta/. Table 3. Monthly variation in total number of bacterial load in pond water recorded during August'93 to July'94 Bacterial load in pond water (CFU/ml) Month Surface water Bottom water 1st phase Aug.'93 3.0lxl0 7 27.72xl0 8 Sep.'93 3.llxl0 7 5.06xl0 8 Oct.'93 l.l9xl0 7 5.9xl0 7 Nov.'93 2.50xl0 7 2.48xl0 7 Dec.'93 1.1 7xl0 7 l.29xl0 8 Jan.'94 l.79xl0 7 l.8lxl0 8 Feb.'94 l.73xl0 7 1.1 lxl0 8 Mar.'94 l.l7xl0 7 l.04xl0 8 Apr.94 l.38xl0 6 l.44xl0 8 May.'94 1.50xl0 5 l.olxl0 6 June.'94 l.27xl0 5 l.lsxw Jul.'94 l.39xl0 5 2.00xl0 6 Data presented are average of four replications The bacterial load in body slime, liver and kidney of fish sample during August'93 to July'94 was found to vary from 0.58xl0 3 to 2.37x 10 7 CFU/g from 0.22xl0 3 to 9.64xl0 6 CFU/g and from 0.15xl0 3 to 9.36xl0 6 CFU/g) respectively during the experimental period. i.e., from August, 93 to July'94 (Table 4).The highest and the lowest bacterial load in all the three organs were found in the month of March'94 and July'94, respectively. Table 4. Monthly variation in total number of bacterial load in fish sample Month lst phase Aug.'93 Sep.'93 Oct.'93 Nov.'93 Dec.'93 Jan.'94 Feb.'94 Mar.'94 Apr.94 May.'94 June.'94 Jul.'94 Bacterial load in fish (CFU/g) Body slime Liver l.06xl0 4 5.78xl0 5 3.67xl0 6 l.32xl0 7 l.l4xl0 7 5.79xl0 6 2.05xl0 7 2.37xl0 7 6.85xl0 5 l.85xl0 5 l.20xl0 3 0.58xl0 3 Data presented are average of 10 fish. 3.35xl0 3 6.74xl0 4 3.88xl0 5 1.48xl0 6 2.96xl0 6 1.44xl0 5 2.66xl0 5 9.64xl0 6 2.37xl0 4 4.58xl0 4 0.23xl0 3 0.22xl0 3 Kidney 2.60xl0 3 5.29xl0 4 9.37xl0 4 4.85xl0 5 2.45xl0 5 5.56xl0 3 2.38xl0 4 9.36xl0 6 5.18xl0 3 3.04xl0 3 O.l8xl0 3 0.15xl0 3 56
Bacteria of C. mriga/a The correlation of different parameters studied from August'93 to J uly'94 indicate that the body length of C. mrigala H. was significantly correlated with body weight, bacterial load in slime was significantly correlated with bacterial load in liver and in kidney and bacterial load in liver was significantly correlated with bacterial load in kidney(tables 5 and 6). Table 5. Correlation between growth parameters and bacterial load in body slime, liver and kidney of Cirrhinus mrigala as observed during the period from August'93 to July'94 Variables r-value (r= 1 0) I. Body length (BL) x Body weight (BW) BL X Bacterial load in slime BL X Bacterial load in liver BL X Bacterial load in kidney 2. BW X Bacterial load in slime BW weight X Bacterial load in liver Body weight X Bacterial load in kidney 3. Bacterial load in slime X Bacterial load in liver Bacterial load in slime X Bacterial load in kidney 4. Bacterial load in slime X Bacterial load in kidney 0.9613*** 0.4939 0.3431 0.3017 0.4783 0.6321 0.3280 0.7245** 0.6487* 0.9596*** *=Significant at 5% level **=Significant at 1% level ***=Significant at 0.1% level The monthly variation in total bacterial load in the pond water, body slime, liver and kidney of the experimental fish, and the existence of significant positive correlation between the bacterial load of body slime and the bacterial load of liver, between the bacterial load of body slime and bacterial load of kidney as well as between bacterial load of liver and bacterial load of kidney, might be due to feed (mustard oil cake, rice bran etc.) used as supplementary feed. Moreover, the month to month fluctuation in the bacterial load might be due to time to time addition of water in the pond. But no significant correlation was found between bacterial load with temperature of pond water. Moreover, the physico- chemical parameter of pond water did not show any significant variation from one month to another. Romanenko (1971) reported that the total bacterial number in reservoir water was 1.43-0.18x10 6 /ml of water. The range of bacterial load in water of the experimental pond as recorded during the present study (l.olxl0 6 /ml to 5.06x10 8 CFU/ml of water) more or less agree with his findings. Horseley (1973) investigated relationship between the bacterial flora of salmon and its environment. He recorded 10 2 to 10 3 bacteria per cm2 of skin and similar number of bacteria found/ ml of water. Chrganowski (1985) reported that the total bacterial population was l.lx10 3 cells m-3 of water in lake Arlington and cell volume was substantially larger in winter than summer and were negatively correlated with temperature which differs from the findings of the present study where higher bacterial load in pond water was recorded in August and September which is probably due to shallowness of the experimental pond that presumably resulted in the existence of favourable condition for the growth of 57
A.N.H. Banu eta/. bacteria in August and September. Tewari and Mishra (1985) reported freshwater bacteria varied from 1 to 3.00x10 3 /ml in the lake water. Lio-Po et al. (1992) reported that total bacterial count CFU/mg) of skin was 1.2x10 3 in apparently normallesioned sample. They also added that the mean CFU/g tissue was 4.3x10 4 in apparently normal specimen. They found that kidney sample likewise revealed the presence of bacteria. Araki and Kitamikadi (1978) pointed out that the population density of bacteria ranged from 0.0 to 1.8x10 5 cell/ml of water in some river and pond waters of Japan. Iqbal (1995) reported that the total bacterial load in pond water, body slime and kidney of fish at Trisal Fish Seed Multiplication Farm, a GoB Farm, varied from 1.3x10 2 to 5.9 x 10 5 CFU/ml of water, 5.4x10 3 to 8.5x10 7 CFU/g of body slime and 0.0 to 2.4x 10 4 CFU/g of kidney, respectively, while that of Jhalak fish farm was 2.0x10 2 to 3.0x10 6 CFU/ml of water, 3.8x10 2 to 2.3x10 8 CFU/g of body slime and 0.0 to 5.3x10 4 CFU/g of kidney which agree with the findings of the present study. The present study revealed that the bacterial population in different substrates were 1.39x 10 5 to 3.11x 10 7 CFU/ml in surface waterl.olx10 6 to 5.90x 10 7 CFU/ml in bottom water, 0.58x10 3 to 2.37 x10 7 CFU/g in body slime, 0.22x10 3 to 9.64 x10 6 CFU/g in liver and 0.15x10 3 to 9.36x 10 6 CFU/g in kidney. The study revealed that there is an existence of positive correlation of bacterial load in body slime with liver as well as with kidney of C. mrigala. References Araki, T. and M. Kitamikadi, 1978. Distribution of common degrading bacteria in aquatic environment. Bull. Jap. Soc. Sci. Fish., 44(10): 1135-1139. Austin, B. and A.M.J. Al-Zaharani, 1988. The effect of anti-microbial compounds on the gastroin~estinal micro-flora of rainbow trout, Salmo gardneri. J. Fish. Biol., 33: 1-14. Charganowski, T.H., 1985. Seasonal abundance and biomass of bacteria in a South Western reservoir,usa. Hydrobiol., 127(2): 117-123. Frazier, W.C. and D.C. Westhoff, 1978. Food Microbiology 3'd ed. McGraw- Hill Book Co. 540pp. Horseley, R.W., 1973. The bacterial flora of the Atlantic Salmon (Salmo salar) L.) in relation to its environment. ]. Appl. Bacteriology, 36: 377-386. Iqbal, M.M., 1995. Bacteriological studies on farmed mrigal, Cin hinus mrigala. M.Sc. Thesis. Department of Fisheries Biology and Limnology, BA U,Mymensingh. Kabata, Z., 1985. Parasites and diseases of fish cultured in the tropics. Taylor and Francis. London and Philadelphia. pp32-39. Lenore, S.C., E.G. Arnold and R.R. Trussel, 1989. Standard Methods for the examination of water and wastewater. APHA- AWWA-WPCF 17'h edition. Lio- Po, G.D., L.J. Albrighr and E.V. Alapide-Tendencia, 1992. Aeromonas hydrophila in the epizootic ulcerative syndrome (EUS) of snakehead, Ophio cephalus striatus and catfish, Clarias batrachus: Quantitative estimation and experimental induction of derma- muscular necrotic lesion. In Disease in Asian Aquaculture. pp: 461-474. Romanenko., V.I., 1971. Total bacterial number in Rybinsk reservoir. Mikrbiol, 40: 707-713. Tewari, B.K. and R. R. Mishra, 1985. Population and productivity of heterotrophic bacteria in fish water lake. Acta. Bot. India, 13(1): 51-56. (Manuscript received 18 March 1999) 58